1. Field of the Invention
This invention relates generally to a transfer mechanism for microorganisms and more specifically, to a device for duplicating a growth pattern of microorganisms.
2. Description of the Prior Art
It is essential in many industries to be able to determine whether or not microorganisms are present in raw materials, finished products and intermediate products used and made in that industry, as well as by-products and effluent resulting from the industrial process, and also to be able to classify the microorganisms if present. Many such microorganisms are not only undesirable but some are detrimental to the health of the consumer of the product and thus prohibited by government regulation.
In addition to the need for detection and quantification of microorganisms in a sample, it is often essential to classify any microorganism so found into its proper taxonomic category.
This need exists for many industries, but it is particularly important for such industries as the drug industry, the food industry and the cosmetic industry. Government regulations in most jurisdications strictly control the quantity of certain microorganisms which is permitted in food, drug, cosmetic and similar products, and accurate tests must be performed at regular intervals to ensure compliance with these regulations.
Several methods exist today for detecting, counting and identifying microorganisms present in a sample. Most methods entail depositing a portion of the sample in or upon one or more culture media which encourage the growth of the microorganisms. Usually, a different kind of culture medium is used for each type of microorganism of interest. Once the microorganisms have grown on these culture media, further tests are often required to complete their identification. This usually requires the transfer of a portion of the growth to other culture media, in order to determine various important characteristics of the microorganisms (such as the ability to ferment certain sugars, or to grow in the presence of certain chemicals).
These methods are widely used in a number of areas including testing of foods, water and effluent for pathogenic bacteria, spoilage organisms, or bacteria indicative of poor sanitary practices, testing of pharmaceutical products, and testing of urine in the diagnosis of urinary tract infections. Other applications include, for example, the control of starter cultures used in fermentation processes (such as in cheese or yoghurt manufacture and in the production of beers and wines).
The invention will be described with respect to the particular application of the food industry, and it is to be understood that it is not restricted thereto, but is equally applicable to any other industry.
Most microorganisms found in foods are saprophytic. Certain of these, when growing in a food, produce chemical changes resulting in food spoilage. Other saprophytes, due to certain of their characteristics, provide information as to the acceptability of the manufacturing process or the hygienic condition of the processing plant. This group of bacteria is usually referred to as the "indicator organisms". Pathogenic organisms can also be found in foods. Certain of these organisms produce toxins or poisons harmful to man; others, such as Salmonella, cause infections. Government regulations limit the quantity of certain indicator organisms in foods, and prohibit the sale of food products containing certain pathogenic bacteria. For example, the presence of Salmonella is specifically prohibited in numerous foods under the Canadian Food and Drugs Act and Regulations, and Section 4(a) of this Statute prohibits the sale of any food that "has in or upon it any poisonous or harmful substance". Other countries have similar prohibitions.
Microbiological examination of foods provides information concerning the quality of the ingredients and the hygienic conditions under which the food was processed. It can also help to determine the effectiveness of any preservative or sterilizing treatment used in the production of the food. The detection of significant levels of indicator organisms, or example, can signal a breakdown in processing plant hygiene. This finding would lead to an examination of equipment and a review of procedures in order to trace the source of the problem. Corrective measures could then be instituted to prevent a reoccurrence.
Microbiological techniques for food examination are similar to those used in other areas of microbiology. For the most part, they consist of cultural procedures, although direct microscopy and serological procedures are used to some extent. The type of examination performed is determined by the type of food product to be examined and, more importantly, by the type of microorganisms being sought. For example, a food being analyzed for mold contamination would be handled differently from one being examined for Salmonella.
A typical procedure to determine a quantity of a specific group of bacteria in a food sample is as follows. A measured quantity of the food is homogenized in a known volume of a suitable diluent. This homogenate is then diluted in a series of ten-fold steps, the number of dilutions being dependent upon the expected level of the bacteria in question in the sample. A measured volume of at least two of these ten-fold homogenate dilutions is then deposited onto a suitable culture medium, contained in Petri Dishes (The "Dilution Petri Dishes"), and evenly spread over the surface of the medium. Usually, two separate Petri Dishes of medium are used for each of the dilutions being transferred. These Dilution Petri Dishes are incubated to allow the organisms to grow. During incubation, each viable bacterial cell should produce a colony on the surface of the culture medium. After incubation, the analyst places each Dilution Petri Dish under a magnifying lens and counts the number of colonies which have formed. Based on this information, and the extent to which the sample was diluted, the analyst is able to deduce the number of organisms present in the original sample that may belong to the group of bacteria in question (known as the presumptive count).
In order to determine whether all of the bacteria included in the presumptive count do, in fact, belong to the required group, one or more confirming tests are required. To this end, the analyst usually transfers up to ten colonies of bacteria from one of the pairs of Dilution Petri Dishes onto fresh culture medium in Petri Dishes, both to verify the purity of the original colony (that is, to determine that only one type of bacterium was present therein) and to increase the number of cells of the bacterium available for the confirming tests. In order to successfully accomplish this transfer, each colony of bacteria must be individually transported using a suitable implement such as a sterile needle. These dishes are incubated, and the colonies that develop on the surface of the culture medium are then used to inoculate one or more culture media, or to perform other tests (such as serology), depending upon the group of bacteria in question. Following the completion of these tests, the analyst evaluates the results obtained for each of the original colonies meeting the confirmation criteria. This fraction is then multiplied by the presumptive count, to yield the confirmed number of bacteria of the group in question in the original sample (known as the confirmed count).
The confirmation procedure described above has a major drawback in that the decision on the part of the analyst as to which colonies to transfer from the Dilution Petri Dishes for further tests is based in large part on subjective criteria. Since such a small proportion of the colonies are carried through the confirmation procedure (often fewer than 5% of the colonies present on the Dilution Petri Dishes), an erroneous decision on even one of the colonies can have a large impact on the final calculation of the confirmed count.
The field of mutation research is another area requiring the transfer or duplication of a large number of colonies. The Ames test for carcinogenicity, for example, tests the ability of a wide variety of ingredients in foods and pharmaceutical products to cause genetic mutations in bacteria (this has been linked to the potential to cause cancer in man). In this test, a bacterium with known growth properties is subjected to the chemical being tested. The bacterium is then allowed to grow and produce colonies on a culture medium (primary medium) in a Petri Dish. Each of these colonies must be transferred onto a series of different culture media (secondary media) in order to detect any changes in growth patterns (for example, the development of resistance to a particular antibiotic to which the bacterium was previously sensitive). The transfer of the bacterial colonies can presently be performed using one of two methods.
The first method involves transferring individual colonies to the secondary media using a sterile needle or other similar device. This method is slow and tedious. It places severe limitations on both the number of culture media that can be included in the experiment and the number of potential cancer-causing chemicals which can be tested. The second method involves stretching a piece of velvet cloth over a solid block (usually of wood) and securing the cloth in place. The surface of the velvet is sterilized by whatever means are feasible. The surface of the velvet is first applied by hand against the colonies growing on the surface of the primary medium and then manually duplicated onto the surfaces of each of the secondary media. This second procedure, if very carefully applied, permits the simultaneous transfer of a larger number of colonies. However, because it is a totally freehand manual operation, significant problems associated with the accurate transfer of colonies are common. These problems will manifest themselves as incomplete transfers due to insufficient pressure or to smearing of colonies due either to excessive pressure or to lateral hand motion while duplicating.
More recently, a novel apparatus for enumerating microorganisms has been developed. As discussed in U.S. Pat. No. 3,929,583 granted on Dec. 30, 1975 to Canadian Patents and Development Limited, this apparatus comprises a membrane filter capable of retaining microorganisms on its surface when a fluid sample is passed through it. A barrier material is imprinted on the surface of the filter which restricts the spread of colonies through its physical properties. The pattern produced defines a plurality of ordered, microbial colony-isolating cells wherein the cells are usually smaller in area than in normal colony area.
The use of the hydrophobic grid membrane filter (hereinafter referred to as HGMF) has produced a substantial advance in the field of microbiology. The regularity in size, shape and optical density, and the orderly arrangement of colonies as a result of the gridded pattern of barrier material of the HGMF has permitted the replacement of manual counting with optoelectronic scanning, thus saving analyst time and producing more reliable and reproducible results.
Even with the advent of the HGMF, the problem still existed as to how to transfer or duplicate the colonies of microorganisms growing on the HGMF to fresh or different culture media so that further tests could be conducted on the microorganisms from the original sample.